It is believed that a significant percentage of proteins exist and carry out their biological function without a well- defined structure. Intrinsically disordered proteins (IDPs) lack a rigid three-dimensional fold or contain large stretches of amino acids devoid of structure. Key to the function of IDPs is their ability to sample a vast array of conformations and to interact with a wide range of protein partners. It is predicted that IDPs are highly prevalent in high order eukaryotic organisms where up to half of their proteins are entirely disordered or contain large stretches of amino acids that are disordered. Further, IDPs are believed to play significant roles in several diseases including cancer, cardiovascular disease, diabetes, and neurodegenerative diseases. While disorder is clearly a trait abundantly used in protein function, it has posed extensive challenges to theoretical and experimental characterization leaving a large knowledge gap in our basic understanding of IDPs. The goal of this proposal is to carry out fundamental thermodynamic and structural studies on a model protein system, E. coli IscU, which utilizes disordered and structured conformations as part of its function. IscU is the scaffold protein for iron-sulfur (Fe-S) cluster biosynthesis and transfer and selectively samples its structured or disordered conformation as it interacts with several protein partners throughout the Fe-S cluster assembly and transfer mechanism. Specific Aim 1 will apply a biophysical toolkit composed of NMR, small- angle X-ray scattering (SAXS), and differential scanning calorimetry to map the structural energetic landscape of IscU. Particularly unique to IscU is that it undergoes cold denaturation at observable temperatures without the aid of chemicals that perturb its energetic landscape. This extremely rare trait will allow us to test and answer structural and thermodynamic questions that still revolve around cold denaturation. Specific Aim 2 will focus on characterizing the structure and energetics of IscU protein-protein complexes. This aim will seek to test if the knowledge of IscU obtained in Specific Aim 1 can be applied in a predictive manner to complicated protein-protein interactions. We will develop methods to test and determine the limits of how well we can build molecular models of IscU's protein-protein complexes using SAXS and carry out thermodynamic studies on IscU's protein-protein interactions. Together, these studies will provide much needed fundamental insights into both the structure and energetics of IDPs and their interactions with protein-partners. Overall, the proposed studies will expose the applicant to the field of protein research and provide training in protein expression and purification, SAXS, and protein NMR methods.